Pulmonary microvascular endothelial cells (PMVECs) possess strongly adherent cell-cell junctions[unreadable] that are necessary to limit fluid, solute and macromolecule permeability into interstitial and alveolar[unreadable] compartments, which is important for efficient gas exchange. PMVEC junction strength is dynamically[unreadable] adjusted by intracellular cAMP concentrations. The type 6 adenylyl cyclase (AC6) synthesizes cAMP at the[unreadable] cell membrane. cAMP signaling is targeted to physiologically relevant effector molecules by type 4[unreadable] phosphodiesterases (PDE4), specifically the -D4 isoform, which is membrane-localized by spectrin. The[unreadable] membrane-localized cAMP pool strengthens PMVEC barrier function. In contrast, Pseudomonas aeruginosa[unreadable] introduces a soluble adenylyl cyclase toxin, ExoY, into PMVECs that generates a cytosolic cAMP pool.[unreadable] cAMP synthesis within the cytosol disrupts, rather than strengthens, the PMVEC barrier. To determine[unreadable] whether membrane or cytosolic AC activity dominates in control of endothelial cell barrier function, we[unreadable] utilized a chimeric mammalian soluble AC enzyme that could be activated by forskolin. Simultaneous[unreadable] stimulation of membrane and cytosolic AC activity by forskolin disrupts, rather than strengthens, the PMVEC[unreadable] barrier, indicating soluble AC activity dominantly controls barrier strength. Preliminary data suggest soluble[unreadable] ACs associate with the centrosome and its associated microtubules, and may therefore reorganize[unreadable] microtubule architecture necessary to induce PMVEC gaps. Thus, this proposal tests the overall hypothesis[unreadable] that membrane-localized ACs produce a cAMP pool that strengthens, whereas cytosolic ACs produce a[unreadable] cAMP pool that disrupts, the PMVEC barrier. Specific Aims test the related Hypotheses that: [1] AC6[unreadable] generates a membrane cAMP pool that is maintained by a spectrin and PDE4(D4) interaction; [2] Soluble[unreadable] ACs generate a cytosolic cAMP pool that controls microtubule organization; and, [3] cAMP that accesses the[unreadable] cytosolic compartment disassembles microtubules and disrupts the endothelial cell barrier. Completion of[unreadable] this work will contribute to our understanding of how cAMP acts to control PMVEC barrier strength, and will[unreadable] seek to resolve pathogenic mechanisms of bacteria like Pseudomonas aeruginosa, which utilize adenylyl[unreadable] cyclase toxins to disrupt the endothelial cell barrier and increase permeability.